DJ-1 is an evolutionarily conserved protein of unknown function. Mutations in the human DJ-1 gene are associated specifically with a form of recessive parkinsonism, suggesting that despite its widespread expression in the body, the major effect of loss of function is neurodegeneration in the brainstem. The current project attempts to understand why this is the case, and we have been following two major strands. One of the well characterized roles of DJ-1 is in responses to oxidative damage. We have shown previously that DJ-1 has an unusual response to oxidation both in vitro and in vivo in that a specific cysteine residue, C106 in human DJ-1, is modified to form a cysteine-sulfinic acid. Replacement of C106 renders DJ-1 non-functional in a number of assays related to cellular survival in the presence of oxidative stress, which we have interpreted to mean that sulfinic acid formation is critical for DJ-1 function. However, a reasonable alternative interpretation is that the cysteine residue itself is critical and the sulfinic acid is an unimportant modification. To address this, we examined the crystal structure of DJ-1 and noted that a nearby residue that is also evolutionarily conserved, Glu18 in human DJ-1, stabilizes the sulfinic acid sidechain of oxidized C106. By manipulation of Glu18, we were able to produce mutant DJ-1 forms that had decreasing ability to support Cys-106 modification. Although the Cysteine residue is present in all of these variants, ability of DJ-1 to protect cells against oxidative-stress induced mitochondrial dysfunction or cell death correlates with the ability to support the modified sulfinic acid sidechain. This indicates that the modification, not the underlying amino acid is critical for function. The next question is to understand why such an unusual protein modification is important. We have previously proposed that DJ-1 can bind RNA in an oxidation-dependent manner, and have shown recently that this extends to the human brain. However, we have not shown that DJ-1 RNA binding is important for its cellular protective function and we are currently exploring this hypothesis by making directed mutations. We are also attempting to understand why mitochondrial function appears to be a major target of DJ-1 activity in cells.